The present invention relates to a novel polynucleotide sequences isolated from tomato and, more particularly, to a novel lycopene cyclase gene and novel control elements controlling its specific expression in chromogenic tissues of plants, e.g., fruit and flower.
Carotenoids--functions and biosynthesis: Carotenoids comprise one of the largest classes of pigments in nature. In photosynthetic organisms carotenoids serve two major functions--as accessory pigments for light harvesting, and as protective agents against photooxidation processes in the photosynthetic apparatus. Another important role of carotenoids in plants, as well as in some animals, is that of providing distinctive pigmentation. Most of the orange, yellow, or red colors found in the flowers, fruits and other organs of many higher plant species are due to accumulation of carotenoids in the cells.
The biosynthesis of carotenoids has been reviewed extensively (Britton, 1988; Sandmann, 1994a). Carotenoids are produced from the general isoprenoid biosynthetic pathway, which in plants takes place in the chloroplasts of photosynthetic tissues and chromoplasts of fruits and flowers.
The first unique step in carotenoid biosynthesis is the head-to-head condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to produce phytoene (FIG. 1). All the subsequent steps in the pathway occur in association with membranes. Four desaturation (dehydrogenation) reactions convert phytoene to lycopene via phytofluene, .zeta.-carotene, and neurosporene, as intermediates. Two cyclization reactions convert lycopene to .beta.-carotene (FIG. 1). Further reactions involve the addition of various oxygen-containing side groups which form the various xanthophyll species (not shown).
It has been established in recent years that four enzymes in plants catalyze the biosynthesis of .beta.-carotene from GGPP: phytoene synthase, phytoene desaturase, .zeta.-carotene desaturase and lycopene cyclase (reviewed in Sandmann, 1994b). All enzymes in the pathway are nuclear encoded.
Genes for phytoene synthase and phytoene desaturase have been previously cloned from tomato (Ray et al., 1992; Pecker et al., 1992).
The red color of ripe tomatoes is provided by lycopene, a linear carotene which accumulates during fruit ripening as membrane-bound crystals in chromoplasts (Laval-Martin et al., 1975). It is presumed to serve as an attractant of predators that eat the fruit and disperse the seeds. Accumulation of lycopene begins at the "breaker" stage of fruit ripening after the fruit has reached the "mature green" stage. In the "breaker" stage, which is indicated by the commencement of color change from green to orange, chlorophyll is degraded and chloroplasts turn into chromoplasts (Gillaspy et al., 1993; Grierson and Schuch, 1993). Total carotenoid concentration increases between 10 to 15-fold during the transition from "mature green" to "red". This change is due mainly to a 300-fold increase in lycopene (Fraser et al., 1994).
The cDNA which encodes lycopene .beta.-cyclase, CrtL-b, was cloned from tomato (Lycopersicon esculentum cv. VF36) and tobacco (Nicotiana tabacum cv. Samsun NN, Pecker et al., 1996, U.S. patent application Ser. No. 08/399,561 and PCT/US96/03044 (WO 96/28014) both are incorporated by reference as if fully set forth herein) and was functionally expressed in Escherichia coli. This enzyme converts lycopene to .beta.-carotene by catalyzing the formation of two .beta.-rings, one at each end of the linear carotene. The enzyme interacts with half of the carotenoid molecule and requires a double bond at the C-7,8 (or C-7,8') position. Inhibition experiments in E. coli indicated that lycopene cyclase is the target site for the inhibitor 2-(4-methylphenoxy)tri-ethylamine hydrochloride (MPTA, Pecker et al., 1996). The primary structure of lycopene cyclase in higher plants is significantly conserved with the enzyme from cyanobacteria but differs from that of the non-photosynthetic bacteria Erwinia (Pecker et al., 1996). Levels of mRNAs of CrtL-b and Pds, which encodes phytoene desaturase, were measured in leaves, flowers and ripening fruits of tomato. In contrast to genes that encode enzymes of early steps in the carotenoid biosynthesis pathway, whose transcription increases during the "breaker" stage of fruit ripening, the level of CrtL-b mRNA decreases at this stage (Pecker et al., 1996). Hence, the accumulation of lycopene in tomato fruits is apparently due to a down-regulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis.
The search for tissue specific control elements in plants is on going, however, only limited number of tissue specific control elements capable of specifically directing gene expression in chromogenic tissues (fruit, flower) have so far been isolated. These include the promoters of the genes E4 and E8 (Montgomery et al., 1993), which are up-regulated by increase in ethylene concentration during tomato fruit ripening, the tomato gene 2A11 gene (Van Haaren and Houck, 1991) and the polygalacturonase (PG) gene (Nicholass et al., 1995; Montgomery et al., 1993), which are upregulated in tomato fruits during ripening.
There is thus a widely recognized need for, and it would be highly advantageous to have, a novel tissue specific control elements capable of specifically directing gene expression in chromogenic tissues.
The search for structural genes encoding enzymes associated with carotenogenesis is ongoing, and every new gene isolated not only provides insight into carotenogenesis, but also provides a tool to control and modify carotenogenesis for commercial purposes (Hirschberg et al. 1997, Cunningham FX Jr. and Gantt B, 1998).
There is thus a widely recognized need for, and it would be highly advantageous to have, a novel lycopene cyclase capable of altering the composition of carotenoids in carotenoids producing organisms.